The hu3F8-BsAb format was designed as a huOKT3 scFv fusion to the C-terminus of the light chain of a human IgG1 (21 (link)). Nucleotide sequences encoding VH and VL domains from our hu3F8, and the OKT3 scFv were synthesized by GenScript with appropriate flanking restriction enzyme sites, and were subcloned into a mammalian expression vector. Two control BsAbs were built on the same platform, Herceptin-huOKT3 and hu3F8-C825 (22 (link)). Linearized plasmid DNA was used to transfect CHO-S cells (Invitrogen) for stable production of BsAb, similar to that described in our previous report (25 (link)). Hu3F8-BsAb titer was determined by ELISA using antigen GD2 and CD3(+) Jurket cell line, and stable clones with highest expression were selected.
The BsAb producer line was cultured in OptiCHO medium and the mature supernatant harvested. A protein A affinity column (GE Healthcare) was used to purify hu3F8-BsAb as previously described (7 (link)), and BsAb was dialyzed into 25 mM sodium citrate, 0.15 M NaCl, pH 8.2 and frozen in aliquots at −80°C. The purity of hu3F8-BsAb was evaluated by both SDS-PAGE (7 (link)), and size-exclusion high-performance liquid chromatography (SE-HPLC).
The BsAb producer line was cultured in OptiCHO medium and the mature supernatant harvested. A protein A affinity column (GE Healthcare) was used to purify hu3F8-BsAb as previously described (7 (link)), and BsAb was dialyzed into 25 mM sodium citrate, 0.15 M NaCl, pH 8.2 and frozen in aliquots at −80°C. The purity of hu3F8-BsAb was evaluated by both SDS-PAGE (7 (link)), and size-exclusion high-performance liquid chromatography (SE-HPLC).
